Nanoparticles have unique properties that have been exploited for use in the delivery of DNA to cells. Metal nanoparticles, such as gold (Au) nanoparticles have been used for DNA delivery because of their low cytotoxicity and ease of functionalization with various ligands of biological significance. In addition to metal nanoparticles, semi-conductor nanoparticles (e.g., quantum dots) (“QD”) within the size range of 3-5 nm have also been used as carriers to deliver molecules into cells. DNA and proteins can be linked to the ligand attached to the QD surface (see, e.g., F. Patolsky et al., J. Am. Chem. Soc. 125, 13918 (2003)).
Nanoparticles have been used to deliver plasmid DNA to a variety of animal cells. It has been found that when DNA coated nanoparticles are incubated with cells not having a cell wall, the cells take up the nanoparticles and begin expressing any genes encoded on the DNA. However, the contemporary plant gene delivery is challenging due to the presence of plant cell walls, which leads to the common reliance on invasive delivery means for genetic transformation of plants. Where nanoparticle-mediated delivery to cells normally having a cell wall is desired, the cell's wall is stripped before the addition of the particles to protoplasts of plant (see, F. Torney et al., Nature Nanotechnol. 2 (2007)). In plant cells, the cell wall stands as a barrier for the delivery of exogenously applied molecules. Many invasive methods, like gene gun (biolistics), microinjection, electroporation, and Agrobacterium, have been employed to achieve gene and small molecule delivery into these walled plant cells, but delivery of proteins has only been achieved by microinjection. Delivery of small molecules and proteins in the presence of a plant cell wall remains unexplored and would be advantageous in order to develop enabling technologies to be deployed in intact plant cell/tissue or organ for in vitro and in vivo manipulations.
Cell penetrating peptides (CPPs) are a novel and fast growing class of short peptides that are known to play an important role in translocation of a wide range of cargo complexes including proteins and DNA across the bio-membranes in mammalian and human cell lines. J. Schwartz and S. Zhang (2000), Peptide-Mediated Cellular Delivery, Curr. Opin. Mol. Ther. 2:162-167; Ü. Langel (2002), Preface in Cell Penetrating Peptides; Processes and Applications, Ü. Langel, editor, CRC Press, Boca Raton; E. Vives and B. Lebleu (2002), The Tat-Derived Cell-Penetrating Peptide in Cell-Penetrating Peptides; Processes and Applications, Ü. Langel, editor, CRC Press, Boca Raton, pp. 3-22. While CPPs have been shown to facilitate cargo delivery in mammalian cells, the use of CPP in plant cells for transfection studies has been limited by a number of factors. A major obstacle to adapting this technology to plants is that, unlike animal cells, plant cells present a dual barrier system (cell wall and plasma membrane) for the internalization of CPPs and their cargos. Therefore, CPPs must overcome these two barriers for efficient translocation. CPPs have been used in plant cells but typically rely on use of permeabilization agents and techniques with the use of CPPs to effectuate delivery of cargo delivery to the plant cells. The HIV-1 TAT protein transduction domain (PTD) is one of the most well studied translocating peptides. Recent reports have shown the potential of TAT-PTD and its oligomers for plasmid delivery by forming a complex with the negatively charged DNA in mammalian cells. I. Ignatovich, E. Dizhe, A. Pavlotskaya, B. Akifiev, S. Burov, S. Orlov and A. Perevozchikov (2003), Complexes of Plasmid DNA with Basic Domain 47-57 of the HIV-1 Tat Protein Are Transferred to Mammalian Cells by Endocytosis-mediated Pathways, J. Biol. Chem. 278:42625-42636; C. Rudolph, C. Plank, J. Lausier, U. Schillinger, R. H. Müller and J. Rosenecker (2003), Oligomers of the Arginine-Rich Motif of the HIV-1 TAT Protein are Capable of Transferring Plasmid DNA into Cells, J. Biol. Chem. 278:11411-11418; Z. Siprashvili, F. Scholl, S. Oliver, A. Adams, C. Contag, P. Wender and P. Khavari (2003), Gene Transfer via Reversible Plasmid Condensation with Cysteine-Flanked, Internally Spaced Arginine-Rich Peptides, Hum. Gene. Ther. 14 (13):1225-33; I. Hellgren, J. Gorman and C. Sylvén (2004), Factors Controlling the Efficiency of Tat-mediated Plasmid DNA Transfer, J. Drug. Target. 12 (1):39-47. Other peptides that have been shown to have translocating properties include pVEC, transportan, penetratin, pep-1 peptides and fragments thereof.
Coating QDs with peptides is one approach that has become popular in the nanoparticle surface engineering for various biotechnological processes. For instance, attachment of cell-penetrating peptides, such as polyarginine and TAT-derived peptides, to the QD surface has allowed translocating QDs into animal cells. Recently, CPPs have become widely used as vehicles for the cellular delivery of molecules in basic and applied biomedical research. With their help, it is possible now to introduce membrane-impermeable substances like peptic nucleic acids (PNA), proteins, oligonucleotides, or nanoparticles into mammalian cells. There is a growing attraction for plant biologists in using CPPs for delivery of biomolecules into the cells and transient expression of the same. Thus, there still remains a need for a method of stable incorporation of genes and other molecules of interest in plants through use of nanoparticle-based delivery.